Metal ions such as iron and copper play an essential role in many cellular processes including energy production, biosynthesis, and antioxidation. The key to their usefulness as enzyme cofactors lies in their ability to participate in electron transfer and to catalyze redox reactions. However, this very chemistry imposes a stringent requirement to tightly regulate the speciation, concentration, and transport of cellular metal ions, since the free ions are themselves highly cytotoxic via Fenton-mediated radical reactions. Copper enters the cell through the transporter CTR1 and is then partitioned between a number of small molecule metallochaperone molecules (CCS, COX17, SCO1, HAH1). These selectively metallate target copper enzymes (e.g., SOD 1, cytochrome c oxidase) or P-type ATPases (MNK, WND), which provide further transport machinery for import of copper into the secretory pathway. Such systems ensure that the concentration of free copper is kept at a negligible level and impart a high degree of selectivity through chaperone-target protein-protein interactions. In this proposal, we develop spectroscopic methods to study the mode of copper binding and transport within a number of these chaperone-target systems, information that has not been forthcoming from X-ray crystallography. The experiments are underpinned by our expertise in X-ray absorption spectroscopy (XAS), the only spectroscopic technique capable of probing the structure of Cu(I) centers in proteins. We also propose to apply novel methods for incorporation of Se as selenocysteine, which will provide us with a unique probe of metal coordination via the Se K XAS of Cuselenocysteine interactions in the chaperones. Using these methods, we plan to determine the modes of copper coordination to the chaperones hCCS and HAH1, and explore the mechanisms of metal transfer between the chaperones and their respective target molecules. The studies will lead to a better understanding of the molecular mechanisms of metal-ion homeostasis and will aid in combating diseases (Menkes, Wilson, ALS) believed to be associated with aberrant metal ion regulation.